Use of proteins to control molluscs

ABSTRACT

The present disclosure includes proteins toxic to Zebra mussels, its method of production, and uses thereof. The protein was isolated from whole cell broth of Pseudomonas protegens CL145A via anion exchange chromatographic fractionation. The protein was found to be a secondary metabolite with highest expression at the fermentative production harvest stage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 15/577,442, filed Nov. 28, 2017, which is aNational Stage of International Application Ser. No. PCT/US2016/033945filed on May 24, 2016, and claims the priority of U.S. ProvisionalApplication Ser. No. 62/167,860 filed on May 28, 2015, the contents ofwhich are incorporated in reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present disclosure relates in general to the field of proteinshaving molluscicidal activity.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

None.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with proteins having molluscicidal. The ability of themussels to quickly colonize new areas, rapidly achieve high densitiesand attach to any hard substratum (e.g., rocks, logs, aquatic plants,shells of native mussels, exoskeletons of crayfish, plastic, concrete,wood, fiberglass, pipes made of iron and polyvinyl chloride and surfacescovered with conventional paints) make it possible for them to causeserious adverse consequences. These consequences include damages ofwater-dependent infrastructure, millions of dollars increase in theoperating expense and significant damage to the ecological systems.

Management of mussels is very important for protecting water-dependentinfrastructure and aquatic ecological systems. There are many proactiveand reactive methods to control and reduce the populations of mussels.Reactive removal includes the mechanical removal, predator removal, andchemical and biochemical removal of adult mussels. For example, fish,birds, crayfish, crabs, leeches and mammals have shown to predatemussels. However, it is unlikely that invasive mussel populations willbe controlled by natural predation, especially in man-made structuressuch as pipes or pumping plants. Proactive measures to control musselsincludes any mechanical, physical or chemical means in which theplanktonic (veliger) mussel life stage is prevented from settling andgrowing into the adult life stage or colonizing on hard substrates.Preventing mussels from colonizing and growing into adult life stages isalso referred to as settlement prevention.

SUMMARY OF THE INVENTION

The present invention includes a method for controlling molluscs in aliquid location comprising administering an effective amount of acomposition having protein that is 80% identical to SEQ ID NO: 1, tocontrol said mollusk at said liquid location. The liquid location can bea body of water or paint, or in pipes filled with liquid.

In one aspect the protein has 90%, 95%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 1.

In another aspect the protein has 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9% sequence identity to SEQ ID NO: 1.

In another aspect, the present disclosure denotes the composition thatcan further comprise gamma-dodecalactone, delta-tridecalactone,piliferolide A, alpha-heptyl-gamma-butyrolactone or11-hydroxy-12-ene-octadecanoic acid.

Yet in another aspect, the said composition further comprises an inertmaterial.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 denotes a plot of the mussel mortality of the cell lysates vs.the days of the mussel assay.

FIG. 2 denotes at least three HMW proteins >260 kDa where expressioncorrelates to the mussel mortality in FIG. 1.

FIG. 3 is a plot of the fraction activity.

FIG. 4 is the SDS-PAGE of the fractions.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As defined herein, “whole broth culture” or “whole cell broth” refers toa liquid culture containing both cells and media. If bacteria are grownon a plate, the cells can be harvested in water or other liquid, wholeculture. The terms “whole broth culture” and “whole cell broth” are usedinterchangeably.

As defined herein, “supernatant” refers to the liquid remaining whencells grown in broth or are harvested in another liquid from an agarplate and are removed by centrifugation, filtration, sedimentation, orother means well known in the art.

As defined herein, “filtrate” refers to liquid from a whole brothculture that has passed through a membrane.

As defined herein, “extract” refers to liquid substance removed fromcells by a solvent (water, detergent, buffer, organic solvent) andseparated from the cells by centrifugation, filtration or other method.

As defined herein, “metabolite” refers to a compound, substance orbyproduct of a fermentation of a microorganism, or supernatant,filtrate, or extract obtained from a microorganism that has pesticidaland particularly, molluscicidal activity.

As defined herein, “carrier” is an inert, organic or inorganic material,with which the active ingredient is mixed or formulated to facilitateits application to plant or other object to be treated, or its storage,transport and/or handling.

As defined herein, “controlling molluscs” means controlling the eggs,larvae, veligers and post-veligers of the molluscs by killing ordisabling them so that they cannot colonize, grow, establish, orreproduce in a given location.

As defined herein, “derived from” and “obtainable from” means directlyisolated or obtained from a particular source or alternatively havingidentifying characteristics of a substance or organism isolated orobtained from a particular source. These terms are used interchangeablythroughout the specification.

As defined herein, an “isolated compound” is essentially free of othercompounds or substances, e.g., at least about 20% pure, preferably atleast about 40% pure, more preferably about 60% pure, even morepreferably about 80% pure, most preferably about 90% pure, and even mostpreferably about 95% pure, as determined by analytical methods,including but not limited to chromatographic methods, electrophoreticmethods. The skilled artisan will recognize that the percentages may beof any incremental value between 20-100%, and each and every valuecovered in that range need not be specifically listed herein.

As defined herein, a “nucleic acid molecule”, is intended to include DNAmolecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

As defined herein, a “vector”, is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

As defined herein, the terms “recombinant host cell” and “transformedhost cell” are used interchangeably and refer to a cell into which arecombinant expression vector and/or an isolated nucleic acid moleculehas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but also tothe progeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The following terms are used to describe the sequence relationshipsbetween a polynucleotide/polypeptide of the present invention with areference polynucleotide/polypeptide: (a) “reference sequence”, (b)“comparison window”, (c) “sequence identity”, and (d) “percentage ofsequence identity”.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison with a polynucleotide/polypeptide of thepresent invention. A reference sequence may be a subset or the entiretyof a specified sequence; for example, as a segment of a full-length cDNAor gene sequence, or the complete cDNA or gene sequence.

As used herein, “comparison window” includes reference to a contiguousand specified segment of a polynucleotide/polypeptide sequence, whereinthe polynucleotide/polypeptide sequence may be compared to a referencesequence and wherein the portion of the polynucleotide/polypeptidesequence in the comparison window may comprise additions or deletions(i.e., gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Generally, the comparison window is at least 20 contiguousnucleotides/amino acids residues in length, and optionally can be 30,40, 50, 100, or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide/polypeptide sequence, a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet et al., Nucleic Acids Research 16:10881-90(1988); Huang et al., Computer Applications in the Biosciences 8:155-65(1992), and Pearson et al., Methods in Molecular Biology 24:307-331(1994).

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See Current Protocolsin Molecular Biology, Chapter 19, Ausubel et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); Altschul et al., J.Mol. Biol., 215:403-410 (1990); and, Altschul et al., Nucleic Acids Res.25:3389-3402 (1997).

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold. These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5877 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequenceswhich may be homopolymeric tracts, short-period repeats, or regionsenriched in one or more amino acids. Such low-complexity regions may bealigned between unrelated proteins even though other regions of theprotein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993))and XNU (Clayerie and States, Comput. Chem., 17:191-201 (1993))low-complexity filters can be employed alone or in combination.

Unless otherwise stated, nucleotide and protein identity/similarityvalues provided herein are calculated using GAP (GCG Version 10) underdefault values.

GAP (Global Alignment Program) can also be used to compare apolynucleotide or polypeptide of the present invention with a referencesequence. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.48:443-453, 1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 100. Thus, for example, the gapcreation and gap extension penalties can each independently be: 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

An effective amount is defined as that quantity of proteins,microorganism cells, supernatant, whole cell broth, filtrate, cellfraction or extract, metabolite and/or compound alone or in combinationwith another pesticidal substance that is sufficient to controlmolluscs. The effective rate can be affected by pest species present,stage of pest growth, pest population density, and environmental factorssuch as temperature, rain, time of day and seasonality. The amount thatwill be within an effective range in a particular instance can bedetermined by laboratory or field tests.

In one embodiment, the protein used in the methods for controllingmolluscs has about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology or sequence identityto SEQ ID NO: 1.

Also provided are nucleic acid molecules that encode said protein SEQ IDNO: 1. These nucleic acid molecules may be DNA, RNA, cDNA, cRNA oranalog sequences. They can be obtained from a Pseudomonas strain or bychemical synthesis or by recombinant methods known in the art.Specifically nucleic acid libraries may be constructed, screened andamplified. For example, a cDNA or genomic library can be screened usinga probe based upon the sequence of a polynucleotide disclosed herein.Probes may be used to hybridize with genomic DNA or cDNA sequences toisolate homologous genes in the same or different plant species. Thoseof skill in the art will appreciate that various degrees of stringencyof hybridization can be employed in the assay; and either thehybridization or the wash medium can be stringent.

The nucleic acids of interest can also be amplified from nucleic acidsamples using amplification techniques. For instance, polymerase chainreaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes. The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

These nucleic acid molecules can be inserted into vectors. The vectorsmay be expression vectors. Recombinant expression vectors containing asequence encoding these nucleic acid molecules are thus provided. Theexpression vector can contain one or more additional polynucleotidesequences, such as but not limited to regulatory sequences, a selectionmarker, a purification tag, or a polyadenylation signal. Such regulatoryelements can include a transcriptional promoter, enhancers, mRNAribosomal binding sites, or sequences that control the termination oftranscription and translation.

Expression vectors, especially mammalian expression vectors, can includeone or more non-transcribed elements, such as an origin of replication,a suitable promoter and enhancer linked to the gene to be expressed,other 5′ or 3′ flanking non-transcribed sequences, 5′ or 3′non-translated sequences (such as necessary ribosome binding sites), apolyadenylation site, splice donor and acceptor sites, recombinationsites, or transcriptional termination sequences. An origin ofreplication that confers the ability to replicate in a specific host mayalso be incorporated.

The vectors may be used to transform any of a wide array of host cellsknown to those of skill in the art. Vectors include without limitation,plasmids, phagemids, cosmids, bacmids, bacterial artificial chromosomes(BACs), yeast artificial chromosomes (YACs), and baculovirus vectors, aswell as other bacterial, eukaryotic, yeast, and viral vectors

The proteins can be obtained, or are obtainable or derived from anorganism having the identifying characteristics of a Pseudomonasspecies, more particularly, from an organism having the identifyingcharacteristics of a strain of Pseudomonas fluorescens or alternativelyfrom an organism having the identifying characteristics of Pseudomonasfluorescens isolate, ATCC 55799 as set forth in U.S. Pat. No. 6,194,194.The methods comprise cultivating these organisms and optionallyobtaining the proteins by isolating these proteins from the cells ofthese organisms.

In particular, the organisms are cultivated in nutrient medium usingmethods known in the art. The organisms can be cultivated by shake flaskcultivation, small scale or large scale fermentation (including but notlimited to continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermenters performed in suitable medium andunder conditions allowing cell growth. The cultivation can take place insuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media maybe available from commercial sources or prepared according to publishedcompositions.

After cultivation, a substantially pure culture or whole cell brothcomprising said strain, or cell fraction, supernatant, filtrate,compound (e.g., metabolite) and/or extract of or derived from saidPseudomonas protegens can be used in formulating a composition of thepresent disclosure. Alternatively, after cultivation, the proteinsand/or metabolites can be extracted from the culture broth.Alternatively, after cultivation, the cells in the pure culture or wholecell broth may be lysed. The lysed cultivation may be unpurified orpurified and used in a formulation for controlling molluscs in a liquid.The purification may vary in degree from removal of cellular debrisand/or nucleic acids, to the isolation of specific classes of compoundsor individual compounds that are used in a formulation for controllingmolluscs in a liquid. In one embodiment, after cultivation, the proteinsand/or metabolites are extracted from the culture broth and are used ina formulation for controlling molluscs. Alternatively, aftercultivation, a substantially pure cell fraction, supernatant, filtrate,and/or extract of or derived from said strain can be used in formulatinga composition for controlling molluscs in a liquid location.Alternatively, after cultivation, one or more proteins and/ormetabolites can be extracted and used in formulating a composition forcontrolling molluscs in a liquid location. The extract can befractionated by chromatography. Chromatographic fractions can be assayedfor toxic activity against, for example, molluscs. This process may berepeated one or more times using the same or different chromatographicmethods.

The proteins set forth above can be formulated in any manner.Non-limiting formulation examples include, but are not limited to,emulsifiable concentrates (EC), wettable powders (WP), soluble liquids(SL), aerosols, ultra-low volume concentrate solutions (ULV), solublepowders (SP), microencapsulation, water dispersed granules, flowables(FL), microemulsions (ME), nano-emulsions (NE), etc. In any formulationdescribed herein, percent of the active ingredient is within a range ofabout 0.01% to 99.99% including each incremental variation in the rangeeven though they are not explicitly listed.

The compositions can be in the form of a liquid, gel or solid. A solidcomposition can be prepared by suspending a solid carrier in a solutionof active ingredient(s) and drying the suspension under mild conditions,such as evaporation at room temperature or vacuum evaporation at 65° C.or lower. A composition can comprise gel-encapsulated activeingredient(s). Such gel-encapsulated materials can be prepared by mixinga gel-forming agent (e.g., gelatin, cellulose, or lignin) with a cultureor suspension of live or inactivated Pseudomonas protegens, or acell-free filtrate or cell fraction of a Pseudomonas protegens cultureor suspension, or a spray- or freeze-dried culture, cell, or cellfraction or in a solution of pesticidal compounds used in the method ofthe invention; and inducing gel formation of the agent.

The composition can additionally comprise a surfactant to be used forthe purpose of emulsification, dispersion, wetting, spreading,integration, disintegration control, stabilization of activeingredients, and improvement of fluidity or rust inhibition. In aparticular embodiment, the surfactant is a non-phytotoxic non-ionicsurfactant which preferably belongs to EPA List 4B. In anotherparticular embodiment, the nonionic surfactant is polyoxyethylene (20)sorbitan monolaurate. The concentration of surfactants may range betweenabout 0.1-35% (including each incremental variation in the range eventhough they are not explicitly listed) of the total formulation; apreferred range is about 5-25% (including each incremental variation inthe range even though they are not explicitly listed)). The choice ofdispersing and emulsifying agents, such as non-ionic, anionic,amphoteric and cationic dispersing and emulsifying agents, and theamount employed is determined by the nature of the composition and theability of the agent to facilitate the dispersion of the proteins of thepresent disclosure.

In another embodiment, the proteins set forth herein can be used incontrolling molluscs, particularly members of the Gastropoda and/orBivalvia classes and more particularly mussels, snails and slugs.

Methods of Use. The proteins of the present disclosure (SEQ ID No. 1)can be used to control molluscs, particularly, a member of theGastropoda and/or Bivalvia class, more particularly mussels (e.g.,Dreissana species) and/or Gastropoda, particularly, snails, whichincludes but is not limited to aquatic snails (e.g., Biomphalariaspecies) and garden snails, including but not limited to brown gardensnails, white garden snails (e.g., Cantareus species, Cornu species,Theba species), and/or slugs, including but not limited to gray gardenslug (e.g., Deroceras sp.), the banded or three-band slug (e.g.,Lehmannia sp.), the tawny slug (e.g., Limacus sp.), and the greenhouseslug (e.g., Milax sp.) in a body of water or on surfaces where molluscssuch as mussels, snails and/or slugs gather or alternatively as ananti-fouling agent in paint. In the event that it is used as anantifouling agent in paint, it is present in an anti-vegetative,biocidally effective amount. Surfaces used by molluscs (such as mussels,snails and/or slugs) include but are not limited to plastic, concrete,wood, fiberglass, pipes made of iron and polyvinyl chloride and surfacescovered with paints and/or coatings. Coatings may be formulated frompigments, binders, additives, and/or carrier fluids and are preferablyapplied in a thin film to provide protection or decoration to a surface.The end product (which contains the active compound) will be used at10-200 mg/L, more specifically at 25-100 mg/L (ppm) or 25-10000 mg/kg(including each incremental variation in the range even though they arenot explicitly listed). It will be applied either as a dry product orsuspended in water into pipes, dam structures, holding tanks, and openwaters such as streams, lakes, irrigation canals, ponds and lakesthrough specific application pumps and mixing systems.

Yet in another embodiment, the present disclosure depicts a compositionincluding other metabolites that have molluscicidal activity. Forexample, lactones and fatty acids as disclosed in U.S. Pat. No.8,968,723.

The present disclosure is also directed to a method comprising a step ofadministering a composition having a protein (e.g., SEQ ID NO: 1), andin combination of an inert material such as clay to enhance the uptakeand hence, mortality of mussels.

Examples of the inert material that may be used in the compositions ofthe present disclosure include, but are not limited to, inorganicminerals such as kaolin, mica, gypsum, phyllosilicates, carbonates,sulfates, or phosphates; or botanical materials such as wood products,cork, powdered corn cobs, rice hulls, peanut hulls and walnut shells. Ina particular embodiment, the inert material can be obtained or derivedfrom a clay mineral (kaolinite, smectite, attapulgite) suspended inwater at a rate of about 1 to 20 mg/liter corresponding to approximately1 to 20 NTU (normalized turbidity units). The inert materials used toenhance mussel siphoning can be applied in solid form or as a suspensionin aqueous solution, preferably water, directly to the water or thelocation (e.g., solid surface) where the mussels are treated. In aparticular embodiment, to enhance product efficacy, an inert materialsuch as clay, silt, sediment or any other material with no nutritionalvalue and with a small enough particle size can be suspended in waterprior to the treatment with a chemical or a biopesticide product.

Conservative Substitutions and Functional Fragments. In comparing aminoacid sequences, residue positions which are not identical can differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. With respect to a reference polypeptide sequence, a testpolypeptide sequence that differs only by conservative substitutions isdenoted a “conservatively substituted variant” of the referencesequence.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are known in the art. Similarly, methods fordetermining protein function are known. For example, the DNA-bindingfunction of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. See Ausubel et al., supra. The ability of a protein to interactwith another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, eithergenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245 246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

Typically, a functional fragment retains at least 50% of the activity orfunction of the polypeptide. In some embodiments, a functional fragmentretains at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 99% or 100% (including each incremental variation in therange even though they are not explicitly listed) of the activity orfunction of the polypeptide.

A functional fragment of a polypeptide can include conservative aminoacid substitutions (with respect to the native polypeptide sequence)that do not substantially alter the activity or function of thepolypeptide. The term “conservative amino acid substitution” refers togrouping of amino acids on the basis of certain common structures and/orproperties. With respect to common structures, amino acids can begrouped into those with non-polar side chains (glycine, alanine, valine,leucine, isoleucine, methionine, proline, phenylalanine and tryptophan),those with uncharged polar side chains (serine, threonine, asparagine,glutamine, tyrosine and cysteine) and those with charged polar sidechains (lysine, arginine, aspartic acid, glutamic acid and histidine). Agroup of amino acids containing aromatic side chains includesphenylalanine, tryptophan and tyrosine. Heterocyclic side chains arepresent in proline, tryptophan and histidine. Within the group of aminoacids containing non-polar side chains, those with short hydrocarbonside chains (glycine, alanine, valine. leucine, isoleucine) can bedistinguished from those with longer, non-hydrocarbon side chains(methionine, proline, phenylalanine, tryptophan). Within the group ofamino acids with charged polar side chains, the acidic amino acids(aspartic acid, glutamic acid) can be distinguished from those withbasic side chains (lysine, arginine and histidine).

A functional method for defining common properties of individual aminoacids is to analyze the normalized frequencies of amino acid changesbetween corresponding proteins of homologous organisms (Schulz, G. E.and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag,1979). According to such analyses, groups of amino acids can be definedin which amino acids within a group are preferentially substituted forone another in homologous proteins, and therefore have similar impact onoverall protein structure (Schulz, G. E. and R. H. Schirmer, supra).According to this type of analysis, conservative amino acidsubstitution” refers to a substitution of one amino acid residue foranother sharing chemical and physical properties of the amino acid sidechain (e.g., charge, size, hydrophobicity/hydrophilicity). Following areexamples of amino acid residues sharing certain chemical and/or physicalproperties:

(i) amino acids containing a charged group, consisting of Glu, Asp, Lys,Arg and His, (ii) amino acids containing a positively-charged group,consisting of Lys, Arg and His, (iii) amino acids containing anegatively-charged group, consisting of Glu and Asp, (iv) amino acidscontaining an aromatic group, consisting of Phe, Tyr and Trp, (v) aminoacids containing a nitrogen ring group, consisting of His and Trp, (vi)amino acids containing a large aliphatic non-polar group, consisting ofVal, Leu and Ile, (vii) amino acids containing a slightly-polar group,consisting of Met and Cys, (viii) amino acids containing a small-residuegroup, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro,(ix) amino acids containing an aliphatic group consisting of Val, Leu,Ile, Met and Cys, and (x) amino acids containing a hydroxyl groupconsisting of Ser and Thr.

Certain “conservative substitutions” may include substitution within thefollowing groups of amino acid residues: gly, ala; val, ile, leu; asp,glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

Thus, as exemplified above, conservative substitutions of amino acidsare known to those of skill in this art and can be made generallywithout altering the biological activity or function of the resultingmolecule. Those of skill in this art also recognize that, in general,single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity. See, e.g.,Watson, et al., “Molecular Biology of the Gene,” 4th Edition, 1987, TheBenjamin/Cummings Pub. Co., Menlo Park, Calif., p. 224.

Polypeptides of the present disclosure encompass those having 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 or more amino acid substitutions compared to anamino acid sequence as set forth in SEQ ID NO: 1, e.g., conservativeamino acid substitutions. Amino acid residues that can be substitutedcan be located at residue positions that are not highly conserved. Theordinarily skilled artisan will appreciate that, based on location ofthe active sites and/or on homology to related proteins, a protein willtolerate substitutions, deletions, and/or insertions at certain of itsamino acid residues, without significant change in its overall physicaland chemical properties.

Polypeptides of the present disclosure encompass those having an aminoacid sequence that is at least 75%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, at least 99.8% or 100% identical to any of the polypeptides shownin SEQ ID NO: 1.

Examples. Sample aliquots of whole cell broth were removed from a 100 Lfermentation of Pseudomonas protegens CL145A at 8, 16, 24.5 and 33 hours(EOF, end of fermentation). The supernatant free whole cell paste waslysed by sonication, and the lysates were examined for activity by livemussel bioassay and analyzed by SDS-PAGE. FIG. 1 is a plot of the musselmortality of the cell lysates vs. the days of the mussel assay.

Mussel activity of the cell lysates is very low over the first 16 hoursof the fermentation and reaches the highest level at the end of thefermentation. SDS-PAGE (FIG. 2) clearly reveals at least three HMWproteins >260 kDa where expression correlates to the mussel mortality inFIG. 1.

The supernatant (SN) from P. protegens EOF cell lysate was fractionatedvia Q-sepharose anion exchange chromatography and the fractions werebioassayed for mussel killing activity. FIG. 3 is a plot of the fractionactivity and FIG. 4 is the SDS-PAGE of the fractions.

The HMW band was excised from an SDS-PAGE gel and submitted to UC Davisproteomics for protein identification via peptide matching. Theresultant protein has the following sequence:

(SEQ ID No. 1) MAFMSKDFTRLLNTLIDQQIKTAGRQTEWFNMSADERAAYIGQVGERLLEMQQSTLSVLAAQHYQMQDNPVSVGDQLQVLQQRRKEMKAIADTPATIAYKQQLDRDILLYSRQDTAISHYDSTWNKALRLLSPGGAKAEVLQADAPAKQKELKGRINRLEKHLSLQVADSTFSQTYVTLFSELQAYKEVSTRYNAWLKAAPQQQAASLDALAKPPRASDELPVNLSLLMMEERPGYIRMNVALVNASTDGREKDFYLEHGRLVVPTDGVLNESFGTAARSLAWQQQYRLKSEPPSMRSPTYAPIRSVLVKTGFVEQYFANHLVSESSLREGFKAQVLSNGRKLLLTGVDRKVPNQVGIQVSGQSPGTSVTREVPLAGALSELINQNADITSFQTLGVEDYRQNSYHPDRDGLFVNIHELERSVGFAEHQYLLEMPQGDAYRSATPFAVMTVEGDKVSSSHLSKAQTETLYQYNAAFFERLEQLRGEGFKASRLFAGSSERATFVQQLTRLLERNHITPAGVLLAQHSRPSLRDIKGNNLNKVLWEQAFAASVWQSHDNDELLFGLGQNLVKNQALSKVLQGGYLQSDIAQAKLLLAPLYEQWRAQAIEMETQRVASANAGQHPGNPKVHVFDQVAVERGLDSKLLNLLLSGPQGLAPADVALRPTVEALLSGDQGRSLRKQALFHALRPVADSFSKAAVPVNAHAALTPKTGADKVMINNRLNQPDPYLILNTHPEQARTDAALLIQDDKYRSYSQFRPDPNNEATRYMSDLDTPFVGGISGTTQTVSNALPELFGSAPSIKQYWQFQMANAAFMIRNGYHSFFETLYVAARYEPQGPDSIGKDLLQTFDRYRAQGRREALHGELYDAVMARVLPIVNQGLAPSEEFHPPRFTDLGPLPALLGQAAKDLQLKTGLASLGAGFEPRQGSADIHQFAADPVQFAQTHTLSAEALVKAGRLPAQGNVQLVEVAPRLYELEYTEHSANSVSGSPDSVPAYFLGYNGPNQANAAPAYVDIPKQARPGSFLFTGTLSGCSLVVTSLDATTYRVYHDGRVNSSLLYDNVVMAVDYKDYQVAGTAEGLAAAYMQVVDGQWQLVFQRQEYQREGQMVWPKLREGAEPLAIQTADSQVQERNRTQFAEYREQVHQNLKKVATQFGVSTEGVADGVYSGGDFSPEHPAIAAWNRLRDAVQAKVSADIEQLGNQRYQLQEQRRGASDKRLIDQQIKQLNLTQDFYRAQYEPVLREAASVEKTWLWQQIQAKQGSAAVVRTDDTAIQGGGDERSTSVGERYAIAEAYQRGARGTAFSDGVRDFREIKIPGLDDKKSALEMKRLFLDGQLTPGQRGALSARISETSQAEYIDKVLRQTATLSEDFRGAGSVFGQLAPQDFYLSLVGDRSGGRCYPLVRAMAVALARGGEAGVNSLVQKLFLASADPQAGSSTLLKNSLIRLHSNVDAVQASKALGQFQLSDVVARLANGSSDSMFALNTQNHSMMVGSTQGPEGRRYYFYDPNVGIFAFDSSKGLAKAMEQHLVRRKLAAHYGSFGSQSQPAFNLVEIDTHKMAEVPVGSGLNVADLSRPEELAGVIGQRRQVEQAVGAQQRVSQDLRLGAALTTFDAEQWGARFDAASTRLAREHQLSSQWIPIIANTEPQPEGGYRVQFINRDQPDQTRWLSTDDGTFVEFRRFVDEHMSVLNEHFTLEHGQIRPRGGVGEVAHVDGLNAGFAVQTLIQWFADKNRKDAAQGVVSPDLATALKIHSYLGLAQIGHGTVQDVAKVTELVQTALRGEALAAESSLKDFASTLGHTVNEGAGVLFGGAMVGLDAYELAHAENDVQKAVFGTQLAFDSASFVSGAAGIGAGLIGASTTAAVLGGAGVILGGLAVGFTALAQAFGAVAEDAKAVGRYFDTLDKAYQGNGYRYDEKQQVLVPLAGAVVKRLDLRSNEVGFDSQYIYRTHHGSTGSGAINYFFWVGDFPRMIHDRAQAIEVRSGIGHGAKPPRLDHGDSRTVILPGTPKSYISYEYMILPGATTRHDTGFDVIRKLEQDRRFDYDFYIFPSEETIRRIHQEYVETPVEVLLDGHNRQLVVPQLPKELYGYLRYDIQGAGGEYLIGLNEGTEVRLSNEAGRQPSRWIIDSSQLESDSITVAKDHLLVGGVKVRLDPAQSGQVLLVNAKGEVRAADFAGQTTYVVKEDASQWQVSGQRIEQHLKELAQAHQLHGQYVVVENYKHGERNVGRAYYEVAKDRMLFTDTDVQQARNAQLGAVIGEHAYFVDAENAAAWRVDIASGKVDAQFAPAFNQSAGQISRFWQEGDAVYLARRYQLKEREAELSFRILGDRMELVGVVGDESLLQLSASNSQHGKDAKTLLNTLLKGYETQATQRDTPVYSLGAPVLEPTAAELITVFGLDNAKVAHRYWVRSSDGIVIKPNLAPPAGQAPRADAPGQAQSAWQIPADLVLAGSQAQPGGQEVFYFYSKAQQVLFRQEGPGQKVLDAGQPSALRLSTPPLANVLNLNGHLLAVTNDGRMARIEATGRLSYEAVNEHWLKAHSNWWKNLAEVAGSNATLAVFGVKAADGKSALPVWYHNGQVVVASSALQGKPLQFLGFDSASASARLFEPESGKLYLQPPLTAQALATAFGKDEVLEASAQLPAAIDWMPKQPLRSAVQVDAGLRLTTVQGEVLLRSNNGDVQLVAVDKGWQQAHLGNLPQALATVAGQWGAKGVLSLQDGDTRGWFDIASGQMFASNGIPGGSDLRFIGVAAGTPNSAYVYSPTAQALYQVKDGKALQLGHYANVERIGSSLLLQGASGNAPQDDLAPPLIAGVDSVVLHGGAGDDTYRFSPAMWAHYRSVVIDNDDPGLALDRVILPVADGKNILVSRRGEDVQLTDTGNGTALVLRQVLGSQAAAHGHLLIELKGDSSMISVEQLLKGFGPSGSAGDSVFELAWSQRETLPAANALSSAADVPDSAADGRGPSLAKLSGAMAAFADTGGAREQLPKNHQAAQAVLVP SLT.

The SEQ ID No. 1 is annotated as cytolysin FitD.

The cytolysin FitD from CL145A (SEQ ID No. 1) can be cloned andexpressed in a non-toxic host for molluscicidal activity and to generateactive protein to determine specific activity in the CL145A cell viamussel bioassay. A gene knockout of the active strain can also revealthe presence of other mussel/insect toxins.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A method for controlling mussels in a liquidlocation comprising: administering an effective amount of a FitD proteinthat is 100% identical to SEQ ID NO:1, to control said mussels at saidliquid location.
 2. The method according to claim 1, wherein said liquidlocation is a body of water or paint.
 3. The method according to claim1, further comprising gamma-dodecalactone, delta-tridecalactone,piliferolide A, alpha-heptyl-gamma-butyrolactone or11-hydroxy-12-ene-octadecanoic acid.
 4. The method according to claim 1,further comprising an inert material.
 5. A composition for controllingmussels in a liquid location comprising: an effective amount of aFitD_protein that is 100% identical to SEQ ID NO:1, to control saidmussels at said liquid location.
 6. The composition according to claim5, further comprising an inert material.